Fig 1: Overexpression of SF3B4 reverses the effects of GPAA1 knockdown on the proliferation, migration and invasion of HCC cells. (A) Protein and (B) mRNA expression levels of SF3B4 after transfection with oe-SF3B4. (C) Protein and (D) mRNA expression levels of GPAA1 after transfection with si-GPAA1 and oe-SF3B4. (E) Protein and (F) mRNA expression levels of GPAA1 after transfection with oe-SF3B4. (G) Protein and (H) mRNA expression levels of GPAA1 after transfection with si-SF3B4#2. (I) Proliferation, (J) colony formation, (K) migration (×100 magnification), (L) invasion (×100 magnification), and (M) MMP2 and MMP9 expression in HCC cells transfected with si-GPAA1 and oe-SF3B4. *P<0.05, **P<0.01 and ***P<0.001 vs. si-NC; #P<0.05, ##P<0.01 and ###P<0.001 vs. si-GPAA1 + oe-NC. GPAA1, glycosylphosphatidylinositol anchor attachment 1; HCC, hepatocellular carcinoma; NC, negative control; oe, overexpression; SF3B4, splicing factor 3B4; si, small interfering.
Fig 2: SF3B4 binds to and stabilizes GPAA1 mRNA. (A and B) It was predicted that SF3B4 could combine with GPAA1, and SF3B4 had a positive correlation with GPAA1 in HCC. (C) Expression of SF3B4 in HCC samples and (D) the overall survival rate of patients with HCC in high SF3B4 level and low SF3B4 level groups. *P<0.05. (E) Binding relationship between SF3B4 and GPAA1 was confirmed by RNA immunoprecipitation. ***P<0.001 vs. IgG antibody. (F) Protein and (G) mRNA expression levels of SF3B4 after transfection with si-SF3B4#1 and si-SF3B4#2. ***P<0.001 vs. si-NC. (H) mRNA stability of GPAA1 following exposure to actinomycin D. ***P<0.001 vs. si-NC. (I) Protein stability of GPAA1 following exposure to actinomycin D. **P<0.01 and ***P<0.001 vs. 0 min. LIHC, liver hepatocellular carcinoma; GPAA1, glycosylphosphatidylinositol anchor attachment 1; HCC, hepatocellular carcinoma; NC, negative control; SF3B4, splicing factor 3B4; si, small interfering.
Fig 3: SF3B4 promotes melanoma cell migration through Talin-1. (A) Immunoblot of B16F10 cells transfected with a control siRNA or two independent siRNAs to SF3B4. (B) Talin-1 protein levels in whole cell lysates from B16F10 cells transfected with a control siRNA or two independent siRNAs to Talin-1. (C) Scratch wound healing assay of B16F10 cells transfected with a control siRNA or two independent siRNAs to SF3B4, with or without Talin-1 knockdown. Statistical analysis was performed using one-way ANOVA with Turkey’s multiple comparisons test. ****p<0.0001.
Fig 4: Knockdown of SF3B4 promotes p21-dependent cellular senescence. (A) Protein levels of p21 and SF3B4 in human fibroblasts transfected with non-targeting siRNA (siControl) or siRNAs against p21 or SF3B4. (B) Representative images of SA-β-gal staining for cells transfected with control, SF3B4 and/or p21 siRNAs as indicated (left) and the percentage of SA-β-gal positive cells (right). Scale bar represents 300 μm. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. Error bars show standard deviation (SD). ****p<0.0001.
Fig 5: Knockdown of SF3B4 induces senescence in human fibroblasts. (A) Protein levels of SF3B4 in human fibroblasts transfected with non-targeting siRNA (siControl) or siRNA against SF3B4. GAPDH served as a loading control. (B) Growth curves of SF3B4 knockdown cells. Statistical significance was calculated using a two-way ANOVA with Sidak’s multiple comparison test. (C) Percentage of cell death in SF3B4 knockdown cells, measured using PI staining. Statistical analysis was based on two-tailed Student’s t-test. (D) Representative images of SA-β-gal staining for control and SF3B4 knockdown cells (left) and the percentage of SA-β-gal positive cells (right). Scale bar represents 300 μm. Statistical analysis was based on two-tailed Student’s t-test. (E) Relative mRNA levels of indicated genes in control and SF3B4 knockdown cells. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test. (F) Protein levels of p21 in control and SF3B4 knockdown cells. GAPDH serves as a loading control. Error bars show standard deviation (SD); ns, not significant, ****p<0.0001.
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